Superantigens are molecules that stimulate, independent of antigen, those T-cells displaying a particular beta chain variable region (Vbeta) of the T-cell receptor. These molecules are the most powerful T-cell mitogens known inducing biological effects at femtomolar concentrations. Recently it has been demonstrated that superantigens possess biological properties, e.g., lethality and emesis, which are separable from their T-cell mitogenicity. Studies have implicated superantigens in such diseases as food poisoning, toxic shock syndrome, scarlet and rheumatic fever, arthritis, multiple sclerosis, diabetes, sudden infant death syndrome, Kawasaki syndrome (a leading cause of acquired heart defects), and MDS. The best characterized superantigens are the microbial toxins from Staphylococcus aureus and Streptococcus pyogenes. The S. aureus toxin, toxic shock syndrome toxin-1 (TSST-1), is the principal causative agent is toxic shock syndrome. The major focus of this proposal is a detailed structural analysis of TSST-1. Initially. this analysis has centered on completing the refinement of native TSST-l in three crystal forms and of a quadruple mutant. These structures are being compared with each other, with staphylococcal enterotoxins C3 and A, and with TSST-1 found in a complex with a class II MHC molecule. Using homology modelling a structural model for S. pyogenes exotoxin A has been produced and models for the other members of this family will be available later this summer. A number of mutants with interesting biological properties, such as reduced superantigenicity or lethality, have been made by our collaborators. Large crystals of six new mutants have recently been obtained and are being analyzed. Structural analysis of well-ordered crystals of staphylococcal exfoliative toxin A (ETA), a nonlethal superantigen, will be performed and the result compared with TSST-l. Guided by the structural data, new mutants, fragments, and chimeras based upon TSST-l will be created to probe biological function. The ultimate goal of this research is to understand the structural foundation for the biological activities for the superantigen family. These activities include not only T-cell mitogenicity but other effects such as host lethality. Such understanding is a key step toward designing new generations of pharmaceutical agents that can block the pathological effects of these molecules or that can modulate the immune system without negative side effects providing new tools in the treatment of autoimmune diseases, cancer and AIDS.